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Interactions of Pyrethroid Insecticides with GABAA and Peripheral-Type Benzodiazepine Receptors

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Interactions of Pyrethroid Insecticides with GABAA and Peripheral-Type Benzodiazepine Receptors AN ABSTRACT OF THE THESIS OF Leslie L. Devaud for the degree of Doctor of Philosophy in Pharmacy presented on August 24, 1988. Title: Interactions of Pyrethroid Insecticides with GABAA and Peripheral-Type Benzodiazepine Receptors Redacted for Privacy Abstract approved: / Thomas F. Murray Pyrethroid insecticides have come into prominent use in recent years due to their high insecticidal activity and reported low mammalian toxicity. This increased use and the development of newer, more potent pyrethroids has made it even more important to elucidate the specific mechanisms of action by which these compounds exert their neurotoxic effects. It is also important to investigate the effects of subtoxic doses of these insecticideson non-target species. The present study focused on the effects of low doses of pyrethroids utilizing both in vivo and in vitromeasures. Pyrethroid insecticides are potent proconvulsants in therat All pyrethroids evincing proconvulsant activity eliciteda similar 25-30% maximal reduction of seizure threshold. The Type II pyrethroids were the most potent proconvulsants with 1RaS,cis cypermethrin having an ED50 value of 6.3 nmol/kg. The proconvulsant activity of both Type I and Type II pyrethroidswas blocked by pretreatment with PK 11195, the peripheral-type benzodiazepine receptor (PTBR) antagonist. In contrast, phenytoin did not antagonize the proconvulsant activity of either deltamethrin or permethrin. Pyrethroids displaced the specific binding of [3H]Ro5-4864 to rat brain membranes with a significant correlation between the log EC50 values for their activities as proconvulsants and the log IC50 values for their inhibition of [3H]Ro5-4864 binding. Both Ro5-4864 and pyrethroid insecticides were found to influence specific [35S]TBPS binding in a GABA-dependentmanner. PK 11195 and the Type II pyrethroid, deltamethrin antagonized the Ro5-4864-induced modulation of [35S]TBPS binding. Pyrethroid insecticides, Ro5-4864 and veratridine influenced GABA-gated 36chloride influx. Moreover, the Type II pyrethroids elicited an increase in 36chloride influx in the absence of GABA- stimulation. Both of these actions were antagonized by PK 11195 and tetrodotoxin. Considered together, these findings suggest that pyrethroid insecticides exert significant effects on mammals at subtoxic doses. They also share complex interactions with ligands for the PTBR ata site allosterically linked to the GABAA receptor. INTERACTIONS OF PYRETHROID INSECTICIDES WITH GABAA AND PERIPHERAL-TYPE BENZODIAZEPINE RECEPTORS by Leslie L. Devaud Neuropharmacology College of Pharmacy Oregon State University Corvallis, OR97331 in partial fulfillment of the requirements for the degree of Doctor of Philosophy completed August 24, 1988 commencement June, 1989 APPROVED: IRedacted for Privacy Dr. Thomas F. Murray, Associate Prgressor of Pharmacy Dr. R chard OhvaT1, Dean of the College of Pharmacy Redacted for Privacy Dean of Graduate S Date thesis is presented: August 24, 1988 For: Leslie L. Devaud ACKNOWLEDGEMENTS Thank you to Elsevier Publications, European Journal of Pharmacology, and ASPET, Journal of Pharmacology and Experimental Therapeutics, for permission to reprint "PK 11195 Antagonism of Pyrethroid-Induced Proconvulsant Activity" and "Involvement of Peripheral-Type Benzodiazepine Receptors in the Proconvulsant Activities of Pyrethroid Insecticides" respectively. Thank you to Dr. Tom Murray in appreciation of his guidance, encouragement, patience, and contagious enthusiasm. A special thank you to my husband, Olivier, and to Ann Blair, Amy Eshleman, Paul Franklin, Mark Leid, Ge Zhang, Libbie Mansell, and the staff at the College of Pharmacy for their invaluable assistance, support and provision of chocolate. And in memory of Dorman J. Hyde (December 1, 1925 December 31, 1987). CONTRIBUTIONS OF OTHER AUTHORS Patricia Szot assisted with the first series of pentylenetetrazol seizure threshold determinations. TABLE OF CONTENTS 1. Introduction 1 2. PK 11195 Antagonism of Pyrethroid-Induced 13 Proconvulsant Activity. 3. Involvement of Peripheral-Type Benzodiazepine 36 Receptors in the Proconvulsant Activities of Pyrethroid Insecticides. 4. Pyrethroid Insecticides and Veratridine Inhibit 74 GABA-Gated 36Chloride Influx into Rat Brain Synaptoneurosomes. 5. Differential Regulation of Specific [35S]TBPS Binding 112 to Rat Brain Membranes by Pyrethroid Insecticides and Ro5-4864. 6. Conclusion. 148 7. References. 151 LIST OF FIGURES 1. Pyrethrins structure. 11 2. Pyrethroids structure. 12 3. Dose response curves for the effects of select 24 Type II pyrethroids on PTZ seizure thresholds in rats. 4. PK 11195 antagonism of the proconvulsant effects of 26 deltamethrin and permethrin. 5. PK 11195 antagonism of the proconvulsant effects of 28 Ro5-4864. 6. Phenytoin effects on the proconvulsant actions of 30 deltamethrin and permethrin. 7. Phenytoin effects on the proconvulsant actions 32 of Ro5-4864. 8. Antagonism of the neurotoxic effects of deltamethrin 34 by PK 11195. 9. Dose response curves for the effects of Type I 58 pyrethroids on PTZ seizure thresholds in rats. 10. PK 11195 antagonism of the proconvulsant effects of 60 cismethrin. 11. Pyrethroid dose-dependent inhibition of specific 62 [3H]Ro5-4864 binding to rat olfactory bulb membranes. 12. Pyrethroid dose-dependent inhibition of specific 64 [3H]Ro5-4864 binding to rat cerebral cortical membranes. 13. Correlation between pyrethroid proconvulsant activity 66 and potency as inhibitors of specific [3H]Ro5-4864 binding in olfactory bulb membranes. 14. Saturation isotherm and Scatchard replot of 68 [3H]Ro5-4864 binding to rat olfactory bulb membranes: effects of deltamethrin. 15. Saturation isotherm and Scatchard replot of 70 [3H]Ro5-4864 binding to rat olfactory bulb membranes: effect of cismethrin. 16. Schild plot of deltamethrin-induced [3H]Ro5-4864 72 affinity shift in rat olfactory bulb membranes. 17. Modulation of GABA-stimulated 36chloride influx by 90 Ro5-4864. 18. Modulation of GABA-stimulated 36chloride influx by 92 the Type II pyrethroid deltamethrin. 19. Ro5-4864-induced inhibition of 50 AM GABA-gated 94 36chloride influx. 20. Inhibition of 50 gM GABA-gated 36chloride influx by 96 the Type II pyrethroid, cypermethrin. 21. Inhibition of 50 AM GABA-gated 36chloride influx by 98 the Type II pyrethroid deltamethrin. 22. Inhibition of 50 AM GABA-gated 36chloride influx by 100 the atypical Type I pyrethroid kadethrin. 23. Inhibition of 50 AM GABA-gated 36chloride influx by 102 the Type I pyrethroid permethrin. 24. Modulation of the deltamethrin-induced inhibition of 104 50 AM GABA-gated 36chloride influx by PK 11195. 25. Antagonism of the deltamethrin-induced inhibition of 106 50 AM GABA-gated 36chloride influx by tetrodotoxin. 26. Antagonism of the veratridine-induced inhibition of 108 50 AM GABA-gated 36chloride influx by tetrodotoxin. 27. Modulation of veratridine-induced inhibition of 50 AM 110 GABA-gated 36chloride influx by PK 11195. 28. GABA-dependency of the Ro5-4864 modulation of specific 130 [35S]TBPSbinding to rat brain membranes. 29. Effect of Ro15-1788 on the Ro5-4864-induced 132 enhancement of specific [35S]TBPS binding. 30. Dose-dependent inhibition of [35S]TBPS binding by 134 PK11195. 31. Effects of PK 11195 on Ro5-4864-induced enhancement 136 of [35S]TBPS binding. 32. Ro5-4864 modulation of the GABA dose-dependent 138 inhibition of [35S]TBPS binding. 33. Deltamethrin antagonism of Ro5-4864-induced 140 enhancement of [35S]TBPS binding. 34. GABA-dependency of deltamethrin-induced inhibition of 142 [35S]TBPS binding. 35. Deltamethrin potentiation of the inhibition of 144 [35S]TBPS binding induced by GABA. 36. Pyrethroid inhibition of specific [35S]TBPS binding 146 to rat cerebral cortical membranes. LIST OF TABLES 1. Effect of PK 11195 on the proconvulsant action of 23 deltamethrin, permethrin and Ro5-4864. 2. Relative potencies of Type I pyrethroids on seizure 55 threshold for PTZ in rats. 3. Relative potencies of pyrethroids and ligands for 56 peripheral- and central-type benzodiazepine receptors as inbititors of specific [3H]Ro5-4864 binding in rat brain membranes. 4. Effects of the Type II pyrethroid deltamethrin and 57 the Type I pyrethroid cismethrin on [3H]Ro5-4864 saturation isotherm binding parameters in rat olfactory bulb membranes. 5. Inhibition of 50 AM GABA-gated chloride influx by 89 pyrethroids and peripheral-type benzodiazepine receptor ligands. 6. Ro15-1788 and PK 11195 sensitivity of the Ro5-4864 127 modulation of specific [35S]TBPS binding. 7. Deltamethrin modulation of the GABA dose-dependent 128 inhibition of specific [35S]TBPS binding. 8. Pyrethroid inhibition of [35S]TBPS binding to rat 129 brain membranes. GABARECEPTOR A GABA Benzodiazepine 4 Agonists: GABA muscimol Agonists: diazepam clonazepam Antagonist: bicuculline Antagonist: Rc15-1788 Inverse Agonist: Ro5-4864/ PK 11195 DMCM Pyrethroid Insecticides Picrotoxinin: TBPS Barbiturates PERIPHERAL-TYPE (MITOCHONDRIAL) BENZODIAZEPINE RECEPTOR a-- tIAgonist: Ro5-4864 Antagonist: PK11195 rproposed Endogenous Ligand: protoporphyrin INTERACTIONS OF PYRETHROID INSECTICIDES WITH GABAA AND PERIPHERAL-TYPE BENZODIAZEPINE RECEPTORS Chapter 1 INTRODUCTION Pyrethroid insecticides have seen increasing use in recent years. As one of the four major classes of neurotoxicant insecticides, pyrethroids have been overshadowed by their more potent and persistent counterparts, the organophosphate, methylcarbamate and chlorinated hydrocarbon insecticides. However, with increasing evidence of undue persistance, high mammalian toxicity, the
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